The present invention relates to the production and screening of libraries of compounds and, more particularly, to the generation and screening of shape and structure libraries produced from large or small size molecules for the purpose of identifying potentially useful agents.
New agents for effectively modulating a range of biological processes have a variety of applications in industry, medicine and agriculture. The identification of structurally unique lead compounds is an important step in selecting such biologically useful agents. Historically and currently, mass screening of collections of large numbers of molecules (chemicals or other compounds) and mixtures of molecules, has been the most successful approach for identifying lead compounds. Most of these collections are either compound databases generated by pharmaceutical research, natural products collections, such as fermentation broths, or more recently, collections of peptides, nucleotides or other synthesized molecules.
Each of these collections, or “libraries”, has its advantages as well as its limitations. Collections generated via research, such as compound databases, can obtain a potentially limitless repertoire of compounds for search (large numbers), however they tend to contain a limited number of diverse structures, representing only a small portion of the total structural diversity possibilities. Natural product libraries can offer structural complexity, however the difficulty in downstream manufacturing of these products, and of reducing leads to useful products is a serious limitation of this type of approach. Peptide libraries are limited to peptides or peptide mimics. There has been limited success in the conversion of peptide chemical leads into pharmaceutically useful drug candidates. These lead compounds are at a disadvantage for generating orally active drug candidates due to the complexity of determining their three dimensional structures for synthesis of small organic molecules and due to the sensitivity of their peptide bonds to acid hydrolysis. However, the structural diversity offered by this technology is its greatest advantage. Nucleotide libraries are also restricted to the genetic repertoire (nucleotides) or nucleotide analogues that preserve specific Watson-Crick pairing and can be copied by a polymerase, hence they are more limited in their useful structural diversity than peptide libraries, however this remains an advantage of these libraries. Nucleotide libraries also offer the capacity for cloning and amplification of DNA sequences, which allows for enrichment by serial selection and provides a facile method for decoding the structure of active molecules.
Compound databases have historically been generated via the chemical modification of existing compounds to generate analogs, which then follow the conventional paradigm of small molecule lead development in which a compound undergoes many rounds of individualized, hand-crafted modification and biological testing en route to drug candidacy. Natural product libraries are derived from collections of natural materials, such as fermentation broths, plant extracts, etc.
Peptide and nucleotide libraries are generated by sequence randomization of individual monomers using a single naturally existing biological linkage (3′-5′ phosphate linkage of nucleotides or amide linkage of peptides). As indicated, the biggest advantage in using peptide and nucleotide libraries is the apparent structural diversity afforded with the technologies. For example, FIG. 1 briefly demonstrates one well known strategy for generating and utilizing “Aptamers”, a library of nucleotide shapes.
For the discovery of drugs and other commercially valuable compounds, small molecule, highly complex libraries containing diverse functionalities have the greatest utility and provide the greatest chance of success. Libraries must also permit identification and evaluation of the structure/activity relationship of the potentially small fraction of active molecules among the larger number of inactive or less active compounds. To satisfy these needs, recent trends are to generate “chemical libraries” and new techniques to evaluate and screen them. “Chemical libraries” have been defined as “intentionally created collection of differing molecules which can be prepared synthetically or biosynthetically”. A type of synthetic strategy which can lead to large chemical libraries is “combinatorial chemistry”. “Combinatorial chemistry” has been defined as “the systematic and repetitive, covalent connection of a set of different ‘building blocks’ of varying structures to each other to yield a large array of diverse molecular entities.” (Gallop, M. A. et al., 1994) Building blocks can include nucleotides, carbohydrates, peptides or peptoids into ordered structures. Chemical libraries generated utilizing combinatorial chemistry can display remarkable diversity. These large libraries can be selected for potential pharmacological activity by their affinity to specified ligands. Several groups have taken advantage of these facts to develop systems utilizing modified and unmodified oligonucleotides and modified and unmodified polypeptides as ligands to bind targets. Many examples are available in the art, a few of which are described herein.
Although oligonucleotides inherently have fewer potential building blocks to provide diversity, they have demonstrated a remarkable affinity for selected targets. Examples include both single-stranded RNA and single- and double-stranded DNA. A great attraction of nucleic acid based combinatorial chemistry is the potential for directed evolution. Repeated cycles of selection for the highest affinity and error-prone PCR can lead to increased diversity and oligomers with an even greater affinity.
The versatility of the binding capabilities of DNA and RNA oligonucleotides seems inexhaustible and the growing number of applications are a tribute to its enormous potential. One of the earliest polynucleotides of this type was directed to the human blood clotting enzyme thrombin (Bock et al., 1992). This study initiated a search for other thrombin inhibitors based on this approach (Bracht and Schroer, 1994; Kubik et al., 1994; Griffin et al., 1993). Numerous other polynucleotide sequences have been selected from initially random libraries of molecules. Examples include DNA and RNA oligomers selected against HIV integrase (Allen et al., 1995), its Rev protein (Giver et al., 1993; Tuerk and MacDougal-Waugh, 1993; Jensen et al., 1994; Jensen et al., 1995; Bartel et al., 1991), and its reverse transcriptase (Chen and Gold, 1994; Tuerk et al., 1992; Schneider et al., 1995), as well as against reverse transcriptase of feline immunodeficiency virus (Chen et al., 1996). Other were developed against human growth factors, such as nerves GF (Binkley et al., 1995), vascular endothelial GF (Jellinek et al., 1994), and basic fibroblast GF (Jellinek et al., 1996; Jellinek et al., 1995) and against Qβ replicase (Brown and Gold, 1995a; Brown and Gold, 1995b). An RNA oligomer has been made against nucleolin (Ghisolfi-Nieto et al., 1996), an essential protein in ribosome biosynthesis. Oligomers against selectins may show potential in treatment of anti-inflammatory diseases (O'Connell et al., 1996). Other proteins against which these oligomers have been selected include immunoglobulin IgE (Wiegand et al., 1996), bacteriophage T4 DNA polymerase (Tuerk and Gold, 1990), bacteriophage R17 coat protein (Schneider et al., 1992), the E. coli rho factor (Schneider et al., 1993), leucine receptive regulatory protein (Cui et al., 1995) and several ribosomal proteins (Dobbelstein and Shenk, 1995; Ringquist et al., 1995).
These polynucleotides can also be selected for their affinity to small molecules. These include early experiments which demonstrated RNA oligomers that bind specifically to a variety of dye molecules (Ellington and Szostak, 1990). Later this finding was extended to DNA oligomers (Ellington and Szostak, 1992). Interestingly, the sequences of these DNA and RNA species are quite distinct, even when selected for the identical substrates.
The oligopeptide substance P, a mammalian neuro-transmitter, was used to select RNA molecules with high affinity against the neurotransmitter by Nieuwlandt et al. (1995). Single amino acids and other small molecules are also able to bind such molecules. Examples include valine (Majerfeld and Yarus, 1994), arginine (Yarus and Majerfeld, 1992; Puglisis et al., 1992; Nolte et al., 1996; Hicke et al., 1989; Geiger et al., 1996; Burgstaller et al., 1995), citrulline (Burgstaller et al., 1995), ATP (Huizinga and Szostak, 1995; Sassanfar and Szostak, 1993), adenosine (Huizinga and Szostak, 1995), D-adenosine (Kluszmann et al., 1996), flavin mono-nucleotide (Fan et al., 1996), theophylline (Jenison et al., 1994), cyanocobalamine (Lorsch and Szostak, 1994).
Another interesting potential of these oligomers was pursued by Morris et al. (1994) who tried unsuccessfully to select for a molecule specific for a reaction transition state, effectively attempting to create a catalyst, Hale and Schimmel (1996), however, did succeed in generating a DNA molecule that induces hydrolysis of a misactivated amino acid bound to a tRNA synthetase, a case of protein synthesis editing. Lorsch and Szostak (1994) succeeded in selecting for several RNA aptamers with 2′ or 5′ polynucleotide kinase activity.
Polynucleotides with modifications of incorporated nucleotides have been selected by Latham et al. (1994), who incorporated 5-(1-pentynyl)-2′-deoxyuridine into thrombin binding DNA molecules. The primary sequence of these modified DNA oligomers was strikingly different from the unmodified DNA molecule.
The use of nucleic acids for therapeutic and diagnostic applications often requires their stability in biological fluids. Aside from chemical modification, nuclease-resistant ligands can be generated by using L-ribose-based nucleotides (Nolte et al. 1996, Klussmann et al. 1996). In this approach the conventional D-RNA directed against the optical mirror image of the target is selected first using repeated rounds of mutation and selection of the nucleic acid and subsequently the corresponding L-RNA is chemically synthesized. L-RNA's with specificity for L-arginine (38-mer, Kd=60 mM, Nolte et al 1996) and D-adensosine (58-mer, Kd=1.7 mM, Klussmann et al. 1996) have been isolated and shown to be stable in human serum at 37° C. Another example includes chirally pure methylphosphonate linkages that are suitable for generating oligomers capable of efficiently hybridizing with DNA or RNA and are highly resistant to metabolic breakdown in biological systems (Reynolds et al. 1996).
Another interesting method for the selection of nucleic acid molecules with highly specific binding to target molecules has been developed and termed “SELEX” (Systematic Evolutions of Ligands by EXponential enrichment), which is described in U.S. Pat. No. 5,270,163 entitled “Nucleic Acid Ligands” and in PCT/US91/04078. SELEX is a method for making a nucleic acid to a desired target molecule involving the selection from a mixture of candidate oligonucleotides and the step-wise iteration of binding, partitioning and amplifying, using the same general selection scheme, to achieve a desired criterion of binding affinity and selectivity. The basic SELEX method has also been modified to achieve a number of specific objectives. (For instance, those described in PCT/US94/10562 filed Sep. 19, 1994, and WO 96/09316 filed Sep. 19, 1995).
For example, SELEX has been used in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA; as a method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photo-crosslinking to and/or photoinactivating a target molecule; in the identification of certain nucleic acid sequences that contain 5-iodouracil residues and that covalently bind to HIV-I Rev protein; in the identification of highly specific nucleic acid ligands able to discriminate between the closely related molecules, theophylline and caffeine; as a method to achieve efficient partitioning between oligonucleotides having high and low affinity for a target molecule; and as a method for covalently linking a nucleic acid to its target.
The SELEX method relies on a process of selection and amplification for enrichment of desired candidate positives from a collection of candidates to identify better candidates or the best candidates from the collection. During the selection part of the process from each parent collection, the bulk binding of the populations of candidates becomes increasingly higher as the sequences are amplified, and those sequences unable to interact with the target are eliminated from the population. Hence, “evolution” of the population occurs due to the increased presence due to amplification of candidates which exhibit the desired activity and the effective elimination of undesirable candidates. Amplification is used to increase the presence of desirable products and to separate those products from those that do not react or have a weaker reaction with a target of interest. (WO 96/09316 entitled “Parallel Selex”).
The Parallel SELEX method describes one potential technique for the identification of DNAs that have facilitating activities as measured by their ability to facilitate formation of a covalent bond between the DNA, including an associated functional unit, and its target. Although this method focuses on the facilitative binding capabilities of DNA, it does not take advantage of the potential for nucleic acids to be evolved in vitro via methods such as “Error-prone PCR” or “Sexual PCR”. The method defines the pool, or collection, of DNAs as being “evolved” due to the enrichment of positives that occurs via an amplification reaction (“exponential enrichment”). The DNA molecules themselves are never evolved.